Regulation of the human beta-like globin genes has been intensively investigated because manipulation of the expression of these genes may permit amelioration of sickle cell disease as well as the beta-thalassemias. The human beta-globin locus is also a classic model of tissue specific gene regulation as well as the developmental regulation of a multigene locus. In recent years there has been a consensus that enhancers upstream of the genes (in the locus control region or LCR) directly interact with the genes and increase their transcription. Using a novel system of reporter gene analysis, however, the applicants have recently demonstrated that a key component of the LCR (5'HS2), as well as other enhancers, does not increase the expression level of a reporter gene to any significant extent, but instead counteracts silencing by repressive chromatin. The investigators have shown that promoter elements can counteract silencing as well as increase transcription; they have also found that characteristics of the integration site strongly influence expression level. The applicants propose that cis-acting transcriptional elements serve primarily to ensure that a gene is expressed in a given lineage, while transcription rate is the product of local chromatin structure and modulation by promoter elements. They also propose a series of experiments to test this hypothesis. The investigators will ask if addition of an enhancer to a locus will stabilize expression; if the proximity of promoter elements to the initiator determines their ability to regulate transcription rate; and if insulators, MARs, and enhancers can alter position effects on expression level. Applicants also will study the coordinate regulation of a model two-gene locus they have developed. All of these experiments will be done in K562 erythroleukemia cells, but they plan to also make ES-derived mice to ask if the 5'HS2 globin enhancer can suppress silencing of gene expression in erythroid cells, and if it can silence expression in non-erythroid cells. These studies may prove useful in the design of gene replacement strategies or drug therapies for the hemoglobinopathies, as well as improving basic knowledge of gene expression.